Photodegradable antimicrobial agents: towards structure optimization

Antibiotic resistance continues to be an ominous threat facing human health globally and urgent action is required to limit the loss of human life. The pollution of antibiotics into the environment is one of the drivers behind the crisis. With this in mind, we have developed novel photodecomposable antimicrobial agents based on an ethanolamine scaffold, which upon photoirradiation decomposes into two major inactive fragments. Herein we describe our further work on the synthesis of novel ethanolamines with a particular focus on structure activity relationship, resulting in four new active compounds which photodecomposed into inactive fragments.


General procedure for the synthesis of the protected anilines
The appropriate aniline (1.0 equiv.) was dissolved in 5 mL of DCM and put in ice bath, then acetyl chloride (1.6 equiv.) was added dropwise followed by the addition of triethylamine (1.6 equiv.). The mixture was stirred for 30 minutes at ambient temperature, then 30 minutes at room temperature. 15 mL of water was added to the mixture, then 15 mL of a saturated solution of NaHCO 3 , then extracted with DCM (3 x 30 mL), and the combined organic phases were dried with MgSO 4 , filtered, and evaporated onto celite and purified by silica-gel flash chromatography.

Synthesis of N-(4-fluorophenyl)acetamide
4-fluoraniline (200 mg, 1.80 mmol) was dissolved in 5 mL of DCM, then acetyl chloride (0.21 mL, 2.88 mmol) was added followed by the addition of triethylamine (0.40 mL, 2.88 mmol) according to the general procedure over 15 min. The reaction mixture was stirred at 0 ºC for 1 h, poured over ice, and extracted with DCM (3 x 15 mL) and the combined organic phases were dried with MgSO 4 , filtered, and concentrated under reduced pressure. 3 It was not possible to purify the products by silica-gel flash chromatography since the reaction gives two nitrated isomers, which retention factors are too narrow, then the next step was performed without further purification.

General procedure for deprotection of the anilines
The appropriate nitrated protected isomers (1 equiv.) were dissolved in MeOH, followed by the addition of HCl 37%. The mixture was refluxed overnight, then concentrated under reduced pressure. Water was added to the mixture then extracted with DCM (3 x 30 mL), and the combined organic phases were dried with MgSO 4 , filtered, and evaporated onto celite and purified by silica-gel flash chromatography.

Synthesis of 2-fluoro-6-nitroaniline and 2-fluoro-4-nitroaniline
The mixture of nitrated isomers (290 mg) was dissolved in 20 mL of MeOH, then 37% hydrochloric acid (2.3 mL) was added and the mixture was refluxed according to the general procedure for 16 h to yield the products

Growth inhibition assay
Compounds 4a-4g, 6a-6r, 8a, and 8b were tested against Staphylococcus aureus ( assay concentration of 50% of the prepared sample, since 50 mL of sample in DMSO/water were added to 50 mL bacterial culture. After adding the samples to the plates, they were incubated overnight at 37 °C and the growth was determined by measuring the optical density at λ = 600 nm (OD600) with a 1420 Multilabel Counter VICTOR3TM (Perkin Elmer, Waltham, MA, USA). A water sample was used as a reference control, growth medium without bacteria was used as a negative control and dilution series of Gentamycin (A2712, Merck, Darmstadt, DE) from 32 to 0.01 mg/mL was used as positive control and visually inspected for bacterial growth.
The positive control was used as a system suitability test and the results of the antimicrobial assay were only considered valid when positive control was passed. The final concentration of DMSO in the assays was ≤ 2% (v/v) and was known to have no effect in the tested bacteria. The data was processed using GraphPad Prism 8.